The proposed project, Directed Lineage Immunizations for Eliciting Broadly Neutralizing Antibody, tests the directed lineage (D/L) approach to HIV vaccination using clade C immunogens. With a D/L vaccine, a series of progressively evolved Env immunogens are used to drive development of a broadly neutralizing antibody (bnAb) response, which is important because of its ability to prevent the establishment of latent reservoirs and viral persistence. No vaccine has yet achieved such a response, but studies of naturally infected humans have shown that in patients in whom a transmitted/founder (T/F) Env has stimulated an unmutated common ancestor (UCA) for bnAb, the co-evolution of virus and B cells can lead to somatic mutations that generate bnAb. The GeoVax directed lineage vaccine is designed to reproduce the series of mutations in Env that led to development of bnAb to the CD4 binding site (CD4bs) in one well studied clade C infected patient, Duke CHAVI patient 505. The vaccines to be tested include four recombinant Modified Vaccinia Ankara (rMVA) viral vaccines, each expressing non-infectious Virus-Like Particles (VLP), and two recombinant gp120 protein vaccines. The MVA-expressed VLPs display Env in a native conformation, while the gp120s are potent at boosting MVA-elicited Ab responses. The vaccines will be produced and then tested in rhesus macaques; use of the rhesus model is essential because the rhesus germline sequence for the UCA for bnAb to the CD4bs is orthologous to the human UCA. Two different regimens will be tested. In the D/L regimen, animals will be immunized with four different rMVA vaccines expressing Env proteins from sequential nodes for the elicitation of bnAb to the CD4bs in patient 505; the rMVA immunizations will be followed by two immunizations with gp120 vaccine corresponding to the most evolved Env. In the T/F regimen, animals will be immunized with rMVAs and gp120s containing only the T/F sequence. Serum samples will be taken from the animals throughout the study, at time points designed to capture peak and contracted responses. The sera will be tested for magnitude, specificity, and breadth of elicited binding antibody (bAb) and neutralizing antibody (nAb). ELISAs will be performed with gp120 and gp160 antigens to demonstrate the overall magnitude and subunit specificity of the immune response and also with antigens specifically designed to quantify immune responses to the CD4 binding site (CD4bs). Testing for nAb will be performed by our collaborator, Dr. David Montefiori. The nAb testing will determine the magnitude and breadth of the nAb response and will determine the CD4bs specificity of elicited nAb. GeoVax will also test elicited Ab for avidity, a potential correlate of protection. Analysis of the data will allow GeoVa to characterize the immune responses and to determine whether the new vaccine design successfully elicited a bnAb response, whether the protein boost increased bnAb titer, and whether the D/L approach improved responses relative to the T/F immunizations. If the D/L is successful at broadening the neutralizing Ab response, it will be advance in clinical testing. If te D/L approach does not broaden the nAb response, the simpler T/F vaccine will be advanced into the clinic.